Project Summary Clostridium perfringens is an important cause of human infections, including traumatic gas gangrene and several common and serious illnesses originating in the intestines, most notably C. perfringens type A food poi- soning, antibiotic-associated diarrhea and enteritis necroticans. Because toxins play a critical role in each of these infections, it is important to understand how this bacterium regulates its toxin production. Towards that goal, our laboratory co-discovered an Agr-like quorum sensing (QS) system in C. perfringens and showed that this QS system regulates production of the toxins (alpha toxin and perfringolysin O) causing gas gangrene. It was later demonstrated by our lab and others that this Agr-like QS system also controls production of all toxins known to mediate enteritis and/or enterotoxemia caused by C. perfringens in humans and livestock. In addition, we showed the Agr-like QS system can be essential for C. perfringens to cause enteric disease. It is unknown whether the Agr-like QS system is similarly important when C. perfringens causes gas gangrene. The C. perfringens Agr-like QS system resembles the paradigm Staphylococcus aureus Agr QS system by involving a signaling peptide (SP), which is processed from a AgrD precursor by the AgrB protein (AgrB also participates in exporting the SP). However, the C. perfringens SP substantially differs in sequence and length from the S. aureus SP. Another key difference is that C. perfringens does not encode a homolog of AgrC, which is the receptor protein that binds SP in S. aureus. It has been suggested that the membrane sensor protein VirS is the SP receptor for the C. perfringens Agr-like QS system but this has not been tested and some evidence suggests VirS may not be a, or the only, SP receptor, at least for some C. perfringens strains. There is currently considerable interest in inhibiting the Agr-like QS system, particularly by interfering with SP binding to its receptor, as an alternative to traditional antibiotics for treating infections by C. perfringens (or other pathogenic clostridia with related Agr-like QS systems). To that end, we and others have identified 3 pro- totype SP-like peptides that inhibit in vitro toxin production regulated by the C. perfringens Agr-like QS system. Considering the above, Aim 1 of this project will elucidate SP:receptor binding during C. perfringens Agr- like QS signaling by testing if fluorescently-labeled SP can bind to VirS by, a) comparing labelled-SP binding levels for wild-type vs. VirS null mutant strains of C. perfringens and b) assessing direct binding of this SP to VirS in vitro. Aim 1 will also evaluate, in an unbiased approach, the possible existence of other (non-VirS) SP receptor proteins using biotin-labeled SP pull-down experiments, followed by mass spectrometry identification of SP binding partners. Aim 2 will then evaluate whether the Agr-like QS system is important when C. perfringens causes gas gangrene. It will also conduct a proof-of-principle test of whether SP-like inhibitor peptides can reduce the ability of C. perfringens to cause disease in animal models.